RIG-I (retinoic-acid inducible gene-I) is a cytosolic, innate immune receptor with the remarkable ability of distinguishing cellular, self RNA from non-self. RIG-I is a complex, multidomain protein consisting of two amino-terminal caspase recruitment domains (CARDs), a helicase domain, and a repressor domain (RD). To understand synergy among the different domains of RIG-I for RNA binding and the contribution of ATP hydrolysis to RIG-I activation, my laboratory determined the structure of the RNA binding portion of human RIG-I (helicase-RD) bound to both dsRNA and a non-hydrolysable ATP analogue, yielding new insights into RIG-I activation (Jiang et al. Nature 2011). RIG-I is principally responsible for detecting uncapped RNAs in the cytoplasm following pathogen entry. Both self and non-self RNAs are characterized the presence of 5-triphosphate (5ppp), but self RNAs are capped by addition of 7-methyl guanosine (m7G) (Cap-0). In higher eukaryotes, the cap is further modified by 2-O-methylation of the 5-end nucleotide ribose (Cap-1). RIG-I is activated by recognition of the uncapped 5ppp of non-self RNAs, allowing the presence of an infectious organism to be signaled via interactions with downstream host factors. In recent work, we have elucidated the molecular mechanisms by which RIG-I can distinguish uncapped viral RNAs from capped, host cell RNAs (Devarkar et al. PNAS 2016). We are continuing structural and functional studies aimed at understanding if RIG-I can discriminate other RNA modifications.